The present invention relates to an assay.
In particular the present invention relates to an assay for use in determining the absence or presence of steroid sulphatase activity.
Steroid precursors, or pro-hormones, having a sulphate group in the 3-position of the steroid nucleus, referred to hereinafter simply as steroid sulphates, are known to play an important part as intermediates in steroid metabolism in the human body. Oestrone sulphate and dehydroepiandrosterone (DHA) sulphate, for example, are known to play an important role as intermediates in the production, in the body, of oestrogens such as oestrone, oestradiol and oestrogenic steroids such as androstenediol.
In particular, and by way of example, oestrone sulphate is known to represent one of the major circulating oestrogen precursors particularly in post-menopausal women and oestrone sulphatase activity in breast tumours is approximately a million fold greater than that of other enzymes involved in oestrogen formation (James et al., Steroids, 50, 269-279 (1987)).
In addition, oestrogens such as oestrone and oestradiol, particularly the over-production thereof, are strongly implicated in malignant conditions, such as breast cancer, see Breast Cancer, Treatment and Prognosis: Ed. R. A. Stoll, pp. 156-172, Blackwell Scientific Publications (1986), and the control of oestrogen production is the specific target of many anti-cancer therapies, both chemotherapy and surgical, e.g. oophorectomy and adrenalectomy.
So far as endocrine therapy is concerned, efforts have so far tended to concentrate on aromatase inhibitors, i.e. compounds which inhibit aromatase activity, which activity is involved in the conversion of androgens such as androstenedione and testosterone to oestrone and oestradiol respectively.
In WO 91/13083, WO 93/05063 and WO 93/05064 are reported methods, including compounds and compositions for use in those methods, for inhibiting or at least reducing steroid sulphatase activity.
In this regard, WO 91/13083 discloses a method of targeting a different point in the oestrogen metabolic pathway, or rather two different points, that is to say the conversion of DHA sulphate and oestrone sulphate to DHA and oestrone, respectively, by steroid sulphatase activity, and using 3-monoalkylthiophosphonate steroid esters as a steroid sulphatase inhibitor, more especially oestrone-3-monomethylthiophosphonate.
WO 93/05063 discloses novel compounds, including pharmaceutically acceptable salts thereof, having steroid sulphatase inhibitory activity. These compounds are the sulphonate and phosphonate esters of polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase activity. In their broadest sense, the novel compounds have the formula shown as FORMULA I in FIG. 1--wherein: R is selected from H, alkyl, cycloalkyl, alkenyl and aryl; X is P or S; Y is --OH when X is P, and .dbd.O when X is S; and the group O-Polycycle represents the residue of a polycyclic alcohol, the sulphate of which is a substrate for enzymes having steroid sulphatase activity.
WO 93/05064 discloses novel compounds, including pharmaceutically acceptable salts thereof, having steroid sulphatase inhibitory activity, and in some cases with extremely high activity levels. These compounds are the sulphamic acid esters of polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase (EC 3.1.6.2) activity, the N-alkyl and N-aryl derivatives of those sulphamic acid esters. In their broadest sense, the novel compounds of WO 93/05064 have the formula shown as FORMULA II in FIG. 2a--wherein: R.sub.1 and R.sub.2 are each independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; and the group --O-- Polycycle represents the residue of a polycyclic alcohol, the sulphate of which is a substrate for enzymes having steroid sulphatase activity (EC 3.1.6.2).
In WO 93/05063 and WO 93/05064 the reference to polycyclic alcohols, the sulphate of which is a substrate for enzymes having steroid sulphatase activity, refers to polycyclic alcohols of the formula shown as FORMULA III in FIG. 2b--which when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37.degree. C., provides a K.sub.m value of less than 50 .mu.moles/l.
The compositions of WO 91/13083, WO 93/05063 and WO 93/05064 are capable of inhibiting steroid sulphatase activity in vitro and in vivo, have improved activity as steroid sulphatase inhibitors both in vitro and in vivo, provide pharmaceutical compositions effective in the treatment of oestrogen dependent tumours, provide pharmaceutical compositions effective in the treatment of breast cancer, enable oestrogen dependent tumours in mammals, especially humans, to be treated and enable breast cancer in mammals and especially in women to be treated.
U.S. Pat. No. 4,150,126 proposes for use as antitumour agents steroid enol esters of the formula shown as FORMULA IV in FIG. 3a--where: PH is a phenyl group, R.sup.1 is (C.sub.2 -C.sub.4) .beta.- or .gamma.-haloalkyl; R.sup.2 is H, lower alkyl, lower alkoxy or halogen; A provides a C.sub.1 -C.sub.4 hydrocarbon chain between --C(O)-- and X; X is O or S; k and m=0 or 1, with the proviso that when m=1 then k=1; and St is a steroid skeleton to which the ester group is attached at the 3-position and adjacent a double bond in the steroid A ring. However no mechanistic explanation is given of the antitumour activity of those compounds.
In U.S. Pat. No. 4,150,126 a brief mention is made that such steroid and esters can be prepared by transesterification inter alia of steroid-3-sulphonates of the formula shown as FORMULA V in FIG. 3b--wherein St is a steroid nucleus as above defined, and R.sup.3 is lower alkyl, optionally containing chloro- or fluoro-substituents, or phenyl, optionally substituted by chloro-, fluoro- or lower alkyl.
However, no examples are given of any such steroid-3-sulphonates for use as intermediates in the preparation of the described steroid enol esters, let alone any suggestion that such steroid-3-sulphonates might themselves inhibit steroid sulphatase activity, and thus be of potential value in the treatment of oestrogen dependent tumours.
There is therefore a need to have a reliable assay to determine whether or not an agent, which may be a compound or a composition, would be effective as a steroid sulphatase inhibitor. Such an assay would enable agents to be screened easily and effectively for potential clinical use.
There is also a need to have a reliable assay to determine whether or not an agent, which may be a compound or a composition, has reduced or eliminated steroid sulphatase activity after the agent has been administered to a subject--e.g. a patient.
Furthermore, the existing procedures to determine the in vivo effectiveness of such agents require harsh invasive techniques such as surgery to inspect the extent of tumour regression. This is clearly unacceptable if the long term or continual action of an agent needs to be monitored.
The present invention seeks to provide a reliable assay to determine the extent of steroid sulphatase inhibition and, furthermore, to overcome the problems associated with the known methods.